Not applicable.
1. Technical Field
Disclosed are methods for protein trans-splicing, use of trans-spliced proteins in gene therapies and gene therapy vectors that encode proteins that trans-splice. In particular, a method for trans-splicing dystrophin and use in gene therapies of recombinant Adeno-Associated Virus (rAAV) particles that encode trans-spliced dystrophin.
2. Description of the Related Art
Protein splicing elements, protein introns, were first discovered in yeast (Kane, P. M., Yamashiro, C. T., Wolczyk, D. F., Neff, N., Goebl, M., and Stevens, T. H. (1990), Protein splicing converts the yeast TFP1 gene product to the 69-kD subunit of the vacuolar H(+)xe2x88x92adenosine triphosphatase, Science 250, 651-657, incorporated herein by reference). Four years later, after six more protein introns had been characterized, they were renamed xe2x80x9cinteinsxe2x80x9d (Perler, F. B., Davis, E. O., Dean, G. E., Gimble, F. S., Jack, W. E., Neff, N., Noren, C. J., Thorner, J., and Belfort, M. (1994), Protein splicing elements: inteins and exteinsxe2x80x94a definition of terms and recommended nomenclature, Nucleic Acids Res. 22, 1125-1127, incorporated herein by reference). Over 100 inteins have since been found in various precursor (host) proteins in a variety of bacterial, archaebacterial and eukaryotic organisms. An intein is defined as a protein sequence which is embedded in-frame within a precursor protein sequence and which is excised during a maturation process called protein splicing. Protein splicing is a post-translational event involving precise excision of the intein sequence and concomitant ligation of the flanking sequences (N- and C-exteins) by a normal peptide bond.
The chemical mechanism of protein splicing was proposed by several groups in 1993 (Wallace, C. J. (1993), The curious case of protein splicing: mechanistic insights suggested by protein semisynthesis, Protein Sci. 2, 697-705; Cooper, A. A., Chen, Y. J., Lindorfer, M. A., and Stevens, T. H. (1993), Protein splicing of the yeast TFP1 intervening protein sequence: a model for self-excision, EMBO J. 12, 2575-2583; Xu, M.-Q., Southworth, M. W., Mersha, F. B., Hornstra, L. J., and Perler, F. B. (1993), In vitro protein splicing of purified precursor and the identification of a branched intermediate, Cell 75, 1371-1377, both of which are incorporated herein by reference) and has since been supported and refined by experimental data (Xu, M.-Q., Comb, D. G., Paulus, H., Noren, C. J., Shao, Y., and Perler, F. B. (1994), Protein splicing: an analysis of the branched intermediate and its resolution by succinimide formation, EMBO J. 13, 5517-5522; Xu, M.-Q. and Perler, F. B, (1996), The mechanism of protein splicing and its modulation by mutation, EMBO J. 15, 5146-5153; both of which are incorporated herein by reference). Briefly, a typical intein folds upon itself, bringing the upstream and downstream splice junctions together to form an active center. Splicing involves an Nxe2x80x94S or an Nxe2x80x94O acyl shift at the splice sites, formation of a branched intermediate, and cyclization of an invariant Asn residue at the C-terminus of the intein to form succinimide, leading to excision of the intein and ligation of the exteins. Amino acid residues that participate directly in the splicing reaction include a nucleophilic amino acid (Cys or Ser), both at the beginning of the intein sequence and at the beginning of the C-extein sequence (Cys, Ser, or Thr), an internal His, and an Asn at the end of the intein sequence. Practical uses of inteins have been made by engineering controllable inteins which undergo controllable protein splicing or cleavage in vitro, including protein trans-splicing by intein fragment reassembly in vitro (Southworth, M. W., Adam, E., Panne, D., Byer, R., Kautz, R., and Perler, F. B. (1998), Control of protein splicing by intein fragment reassembly, EMBO J. 17, 918-926; Mills, K. V., Lew, B. M., Jiang, S.-Q., and Paulus, H. (1998), Protein splicing in trans by purified N- and C-terminal fragments of the Mycobacterium tuberculosis RecA intein, Proc. Natl. Acad. Sci. U.S.A. 95, 3543-3548; both of which are incorporated herein by reference). In vivo protein trans-splicing has also been shown through intein engineering (Shingledecker, K., Jiang, S.-Q., and Paulus, H. (1998), Molecular dissection of the Mycobacterium tuberculosis RecA intein: design of a minimal intein of a trans-splicing system involving two intein fragments, Gene 207, 187-195; Wu, H., Xu, M.-Q., and Liu, X.-Q (1998b), Protein trans-splicing and functional mini-inteins of a cyanobacterial DnaB intein, Biochim. Biophys. Acta 1387, 422-432; each of which are incorporated herein by reference) and the discovery of a naturally occurring trans-splicing intein (Wu, H., Hu, Z., and Liu, X.-Q. (1998a), Protein trans-splicing by a split intein encoded in a split DnaE gene of synechocystis sp. PCC6803, Proc. Natl. Acad. Sci. U.S.A. 95, 9226-9231; incorporated herein by reference). But until now, no practical use has been described for spontaneous or automatic in vivo protein trans-splicing.
Duchenne muscular dystrophy (DMD) is the most common form of X-linked muscular dystrophy, with a world-wide incidence of one in 3,500 male births (Emery, A. E. H., Duchenne Muscular Dystrophy, Oxford University Press, 1993: 392; incorporated herein by reference). DMD patients appear normal until 3-5 years of age, when they begin to experience progressive muscular weakness, starting with large proximal skeletal muscles. The typical affected individual is wheelchair-bound by the age of 12 and succumbs to cardiac or respiratory failure in the mid to late 20s. Becker muscular dystrophy (BMD) is a milder form with delayed onset and longer life span. Most DMD/BMD cases are transmitted via an unaffected mother (heterozygote), whereas 30% of cases have no previous family history and are considered to be due to a de novo mutation in the germ line of either the mother or her parents.
DMD and BMD are caused by a defective dystrophin protein in a patient""s muscle cells (See, Straub, V. and Campbell, K. P. (1997), Muscular dystrophies and the dystrophin-glycoprotein complex, Curr. Opin. Neurol. 10, 168-175; Brown, Jr., R. H. (1997), Dystrophin-associated proteins and the muscular dystrophies, Ann. Rev. Med. 48, 457-466; Michalak, M. and Opas, M. (1997), Functions of dystrophin and dystrophin associated proteins, Curr. Opin. Neurol. 10, 436-442, for recent reviews; each of which are incorporated herein by reference). Dystrophin is a large protein of 3,685 aa and has three structurally distinct regions. The N-terminal region is 136 aa long and forms a globular domain. The C-terminal region is 645 aa long and forms a second globular domain. The central region is a long and rod-like domain that consists of 24 repeats of a triple helical coiled-coil, or of 9 repeats in the smaller, but still functional, Becker form. The N- and C-terminal domains are separated, both in primary sequence and in tertiary structure by the central region. Each repeat is approximately 109 aa long, and there is 10-25% sequence identity between repeats. Each individual repeat is believed to fold independently into a structural module, and neighboring repeats are connected by a short, flexible linker sequence.
Dystrophin is a part of the dystrophin-glycoprotein complex and is thought to function by forming a submembrane lattice which enhances the tensile strength of the muscle membrane and by serving as an anchor for membrane proteins. The human dystrophin gene was identified in 1986 (Monaco, A. P., Neve, R. L., Colletti-Feener, C., Bertelson, C. J., Kurnit, D. M., and Kunkel, L. M. (1986), Isolation of candidate cDNAs for portions of the Duchenne muscular dystrophy gene, Nature 323, 646-650; incorporated herein by reference), the dystrophin protein was identified in 1987 (Hoffman, E. P., Brown, Jr., R. H., and Kunkel, L. M. (1987), Dystrophin: the protein product of the Duchenne muscular dystrophy locus, Cell 51, 919-928; incorporated herein by reference), and the complete dystrophin gene sequence (a 14-kbp cDNA) was cloned and determined by 1988 (Koenig, M., Hoffman, E. P., Bertelson, C. J., Monaco, A. P., Feener, C., and Kunkel, L. M. (1987), Complete cloning of the Duchenne muscular dystrophy (DMD) cDNA and preliminary genomic organization of the DMD gene in normal and affected individuals, Cell 50, 509-517; Koenig, M., Monaco, A. P., and Kunkel, L. M. (1988), The complete sequence of dystrophin predicts a rod-shaped cytoskeletal protein, Cell 53, 219-226; both of which are incorporated herein by reference). Most (over 65%) of the defective dystrophin genes found in DMD and BMD patients exhibit the loss (deletion) of one or more exons, with two deletion hot spots located in the 5xe2x80x2 end region and in the central region of the gene, resulting in smaller dystrophin protein. Deletions that cause frameshifts usually lead to DMD and are predicted to produce either a severely truncated dystrophin or no dystrophin at all.
Progression of the DMD and BMD disease cannot yet be slowed by therapeutic treatment. At present glucocorticoid administration is the only drug therapy, but it is only partially effective and has frequent side-effects.
Alternative methods are being sought but have not yet been found. One potential approach is to upregulate the utrophin gene that encodes a close molecular analog of dystrophin (Karpati, G., Gilbert, R., Petrof. B. J., and Nalbantoglu, J. (1997), Gene therapy research for Duchenne and Becker muscular dystrophies, Curr. Opin. Neurol. 10, 430-435; Gramolini, A. O., Angus, L. M., Schaeffer, L., Burton, E. A., Tinsley, J. M., Davies, K. E., Changeux, J. P., and Jasmin, B. J. (1999), Induction of utrophin gene expression by heregulin in skeletal muscle cells: role of the N-box motif and GA binding protein, Proc. Natl. Acad. Sci. U.S.A. 96, 3223-3227; both of which are incorporated herein by reference). However, it is impossible to predict if or when a nontoxic pharmacological agent would be identified to upregulate the human utrophin gene. Myoblast transplantation offers another potential treatment, but its clinical application has several limitations, including immunological problems, low spread and poor survival of the transplanted myoblasts (Qu, Z., Balkir, L., van Duetekon, J. C., Robbins, P. D., Prochnic, R., and Huard, J. (1998), Development of approaches to improve cell survival in myoblast transfer therapy, J. Cell Biol. 142, 1257-1267; Moisset, P. A., Gagnon, Y., Karpati, G., and Tremblay, J. P. (1998a), Expression of human dystrophin following the transplation of genetically modified mdx myoblasts, Gene Ther. 5, 1340-1346; Moisset, P. A., Skuk, D., Asselin, I., Goulet, M., Roy, B., Karpati, G., and Tremblay, J. P. (1998b), Successful transplantation of genetically corrected DMD myoblasts following ex vivo transduction with the dystrophin minigene, Biochem. Biophys. Res. Commun. 247, 94-99; Miller, R. G., Sharma, K. R., Pavlath, G. K., Gussoni, E., Mynhier, M., Lanctot, A. M., Greco, C. M., Steinman, L., and Blau, H. M. (1997), Myoblast implantation in Duchenne muscular dystrophy: the San Francisco study, Muscle Nerve 20, 469-478; each of which are incorporated herein by reference). Another possibility is to convert DMD to BMD by correcting the frameshift mutation in the DMD dystrophin gene at the RNA level (Matsuo, M. (1996), Duchenne/Becker muscular dystrophy: from molecular diagnosis to gene therapy, Brain and Development 18, 167-172; incorporated herein by reference), but the necessary molecular tools remain to be found.
Gene therapy is recognized as the most plausible candidate for an effective therapy for DMD and BMD (reviewed in Karpati et al., 1997). A simple and proven way is to transfer into patient muscle cells a functional dystrophin gene that produces functional dystrophin. The endogenous defective dystrophin gene is harmless in the presence of the transferred functional dystrophin gene because DMD/BMD are recessive. A miniature version of the dystrophin gene, which was isolated from a BMD patient with very mild symptoms (England, S. B., Nicholson, L. V. B., Johnson, M. A., Forrest, S. M., Love, D. R., Zubrzycka-Gaarn, E. E., Bulman, D. E., Harris, J. B., and Davies, K. E. (1990), Very mild muscular dystrophy associated with the deletion of 46% of dystrophin, Nature 343, 180-182; Love, D. R., Flint, T. J., Sally, A. G., Middleton-Price, H. R., and Davies, K. E. (1991), Becker muscular dystrophy patient with a large intragenic dystrophin deletion: implications for functional minigenes and gene therapy, J. Med. Genet. 28, 860-864; both of which are incorporated herein by reference), can be used in the gene transfer. This minigene (6 kbp cDNA) produces a smaller (1,983 aa, 200 kDa) but still functional dystrophin, which is only slightly less effective than the full length dystrophin gene when tested in transgenic mdx mice. In efficiently transduced muscles of mdx mice, loss of force induced by lengthening contractions was alleviated.
Until now adenovirus-based vector has been the most efficient vector for dystrophin gene transfer into muscles when tested on animal models (mdx mouse and dog) at an early age (see, Karpati et al., 1997 for a review). However, adenovirus vectors have several important drawbacks. First, cellular and humoral immunity triggered by leaky expression of adenovirus proteins and input load of adenovirus proteins, respectively, eliminates transduced fibers and may enhance cytotoxic effects. Second, early non-immunological toxic effects reduce muscle force by xcx9c20% and restrict the maximum adenovirus titer that can be used. Third, there is limited uptake of adenovirus into mature muscle fibers (compared to myoblasts), probably due to lack of cell receptors and the basal lamina barrier. Fourth, when directly injected into a limb muscle, adenovirus vector shows a moderate spread (1-3 mm), which necessitates a high concentration of injection sites. Adenovirus vector injected into the systemic venous circulation is mostly expressed in liver. New adenovirus vectors that are under development may partially overcome some of these drawbacks, but progress is limited.
Adeno-associated Virus (AAV) vector has characteristics that overcome the drawbacks associated with adenovirus vector. First, there are no deleterious CTL immune reactions. Second, AAV vector has less non-immunological toxic effects. Third, AAV vector efficiently transduces mature muscle fibers. Fourth, AAV vector can be prepared free of contaminating adenovirus because of the large size difference between the virus particles. Fifth, AAV itself has no known pathogenicity. Sixth, when directly injected into muscle, AAV vector diffuses more broadly and rapidly due to its smaller particle size and due to its ability to bypass myofiber basal laminae and thereby transduce mature muscle cells. In addition, AAV vector can integrate into the host genome, even in quiescent cells, improving the longevity of the transgene.
Recombinant AAV vectors harboring test genes such as LacZ are capable of achieving highly efficient and sustained gene transfer in the mature muscle of immuno-competent animals for more than 1.5 years without detectable toxicity. Vector integration into the host DNA and the lack of CTL response against transduced cells support the potential of rAAV vectors for genetic muscle diseases. Recently, researchers have greatly improved their ability to generate high titer and high quality rAAV. Successes have been reported in using rAAV vectors to deliver numerous reporter genes as well as therapeutic genes for metabolic diseases, including producing secreted therapeutic proteins using muscle as a platform (Li, J. Dressman, D., Tsao, Y. P., Sakamoto, A., Hoffman, E. P., and Xiao, X., rAAv Vector-mediated sarcoglycan gene transfer in a hamster model for limb girdle Muscular Dystrophy, Gene Therapy (1999) 6:74-82; Greelish, J. P., Su, L. T., Lankford, E. B., Burkman, J. M., Chen, H., Konig, S. K., Mercier, I. M., Desjardins, P. R., Mitchell, M. A., Zheng, X. G., Leferovich, J., Gao, G. P., Balice-Gordon, R. J., Wilson, J. M., and Stedman, H. H. (1999), Stable restoration of the sarcoglycan complex in dystrophic muscle perfused with histamine and a recombinant adeno-associated viral vector, Nat. Med. 5, 439-43; both of which are incorporated herein by reference). Yet using rAAV vector to treat genetic muscle diseases is just beginning to be explored.
The effectiveness of rAAV vectors has been demonstrated in a gene therapy for limb girdle muscular dystrophy (LGMD) 2F, which is caused by mutations in the xcex4-sarcoglycan (SG) gene. A rAAV vector was used for genetic and biochemical rescue in the Bio 14.6 hamster, a homologous animal model for LGMD 2F (Li et al., 1999). Subsequently, efficient and long-term xcex4-SG expression was demonstrated, accompanied by nearly complete recovery of physiological function deficits, after a single dose rAAV vector injection into the tibialis anterior (TA) muscle of the dystrophic hamsters. Recombinant AAV vector treatment led to more than 97% recovery in muscle strength for both specific twitch force and specific tetanic force, when compared to the age-matched control. Vector treatment also prevented pathological muscle hypertrophy, and resulted in normal muscle weight and size. Finally, the histopathology of vector treated muscle showed substantial improvement. This is the first evidence of a successful functional rescue of an entire muscle after AAV mediated gene delivery. These results demonstrate the feasibility of in vivo gene therapy for dystrophic patients using rAAV vectors.
Recombinant AAV vector, until now, has been excluded from gene therapy for DMD and BMD because its maximum insert gene size (4.5-4.8 kbp) is smaller than the size of a functional dystrophin gene or minigene. For example, a vector containing the Becker cDNA must accommodate a sequence of about 7 kbps, the 6 kbp coding sequence plus approximately 1 kbp of necessary accessory sequences. A method is therefore desired which solves this problem and allows the use of AAV vector in gene therapy for DMD and BMD and for other therapies which require the transfer of other nucleotide sequences of sizes greater than the packaging limits of a given vector.
A method is provided that makes use of spontaneous in vivo protein trans-splicing to circumvent packaging limitations of gene therapy vectors, particularly rAAV. The method includes the steps of 1) breaking a gene, for instance the functional dystrophin minigene, into at last two pieces, both of which are smaller than the maximum insert gene size of a transfer vehicle, 2) modifying each piece by operably linking an appropriate fragment of an intein coding sequence and suitable genetic regulatory elements to the dystrophin coding sequences to form extein genes, and 3) transferring each of the two or more extein genes into target cells by using an appropriate vehicle, such as rAAV. The two or more extein genes, once transferred into the same target cell, will produce two or more exteins that will automatically splice together to form a functional dystrophin protein.
More broadly, a method is provided for trans-splicing proteins, including the steps of providing two or more nucleic acids, each encoding and capable of expressing protein fragments to be joined at a junction site by trans-splicing. Preferably, the two or more nucleic acids are produced by severing, at a junction site, a nucleotide sequence encoding a single protein a gene product. Flanking each junction site are nucleotide sequences encoding relative N- and C-extein segments. Relative to each junction site, the 3xe2x80x2 end of the nucleic acid encoding the N-extein segment includes, in phase, a sequence encoding an N-terminal portion of a split intein. The 5xe2x80x2 end of the nucleic acid encoding the relative C-extein segment having, in phase, a sequence encoding a C-terminal portion of the same split intein. A split intein can be a native split intein or a split intein engineered from a native intein, such as the Ssp DnaE and Ssp DnaB inteins, or an engineered split intein prepared synthetically with reference to known intein consensus sequences and structures.
Three or more protein or peptide sequences can be joined at two or more junction sites. In such a case, a different intein is utilized at each junction site to ensure correct assembly of the protein by preventing cross-splicing of the extein fragments.
Also provided is a nucleic acid including at least one of the above-described extein genes.
In one application, the extein genes are transferred to a recipient, such as a human patient or to an in vitro or ex vivo cell culture. The extein genes are transferred by a vehicle, which is preferably a recombinant virus particle, and most preferably a rAAV particle. The particles can be administered either as a population of individual extein genes, or collectively, as a population of particles representing an extein gene set. The particles can be, therefore, administered as a pharmaceutical composition that minimally includes a nucleic acid encoding one of an extein gene, a vehicle containing a nucleic acid encoding an extein gene, the product of an extein gene or the spliced product of two or more extein gene products, and a suitable excipient. The excipient is chosen as suitable for, and preferably optimal for, a chosen delivery route such as parenteral delivery, including intramuscular delivery, or delivery to an in vitro or ex vivo cell culture. The extein gene pharmaceutical composition, or the trans-spliced product thereof, can be packaged as a kit that includes the pharmaceutical composition packaged within a suitable sealed vessel or container.
In one embodiment, the transferred nucleotide sequences encode human dystrophin, or a functional derivative, homolog or analog thereof and the viral vector is AAV. Recombinant AAV particles include a dystrophin extein gene, including at least one partial coding sequence of the dystrophin gene flanked in phase by a coding sequence of a C- and/or N-terminal portion of an intein and under transcriptional control of a suitable promoter and terminator. When expressed in a cell, along with additional extein genes which represent, collectively, an extein gene set including a full complement of dystrophin extein genes, the extein protein segments assemble to form an intact dystrophin protein. Other well-characterized and promising candidates for use in the methods and compositions described herein are the Factor VIII, dysferlin and ATP binding cassette transporter genes.